Rev. Bot., Vol. VI, Nr. 1(8), Chişinău, 2014 103

THE COMPLEX RESEARCH OF INTRODUCTION OF GIGANTEA HOOK T. P. Mazur, N. V. Nuzhyna, A. Ya. Didukh The Botanical garden named after O. V. Fomin of ESC “Institute of biology” of Taras Shevchenko National University of Kyiv. S. Petlyury (Cominterna) 1 Str, Kyiv 01032, Ukraine.

Abstract. The complex research of Nymphaea gigantea Hook plants introducing in greenhouse of water and coastal-water plants of Botanical garden named after O. V. Fomin was done. The peculiarities in structure of floating and above-water leaves of a were proved by anatomical researches. Key words: introduction, greenhouse, nymphaea gigantea. INTRODUCTION Among the floral biodiversity the water plants and their groups get the most anthropogenic influence and vanish because they are rather sensitive indicators of the condition of the aquatic environment. That is why most of them are included to the “Red lists” or “Red books” of the different regions of the world. The role of water plants in a biogeochemical cycle of matter and energy, in the process of self-purification of ponds is a great one and it is difficult to overestimate it. Water plants less depend on climate conditions as compared to ground-based plants that is why relic, rare genera and species among them have remained for present days. The development of Salisb. family members goes back to Cretaceous period. One of the ways of protecting the plants of Nymphaea L genus is their introduction in the conditions of protected soil. The works on introduction and protection the members Nymphaeaceae family have been carrying out for 40 years in Botanical garden named after O. V. Fomin. The important stage for positive results is a complex studying of bioecological peculiarities of the species and their ontogenesis in conditions of ex situ.

MATERIALS AND METHODS The object of our research were Nymphaea gigantea Hook plants taken from the collection of the Botanical garden named after O. V. Fomin of Taras Shevchenko National University of Kyiv. The seeds of N. gigantea were received in 1986 from 104 Rev. Bot., Vol. VI, Nr. 1(8), Chişinău, 2014

St. Peterburg (Russia) Botanical Garden of BIN, their inventory number is 80586. For more detailed studying of the plants of this genus the methodology of modeling the control conditions of existence (similar to natural conditions) in artificial ponds in greenhouse conditions of the Botanical garden was used. The systematical analysis of the Nymphaeaceae family representatives was experimented on the systems of A. L. Takhtadzhian [9], R. K. Brummitt [11]. The biomorphological peculiarities of the plants were described on the basis of E. G. Serebryakov’s [8], O. Е. Vasiliev’s [2], F. Henkel’s, F. Rehnelt’s, L. Ditman’s works [14]. The periods of ontogenesis were studied on the basis of T. A. Rabotnov’s [7], А. А. Zhukova’s works [3]. The colours of plant’s organs were observed with the scale of the colours of А. S. Bondartsev [1]. The characteristic of the climate conditions of the natural distribution was composed on the basis of scientific literature, such as: S. Hejný [12; 13], D. Х. Campbel [4], A. L. Takhtadzhian [10] and M. E. L’vovych [5]. For anatomical studies, the floating and above-water leaves of N. gigantea, collected in the phase of 2–3 days after disclosure were used. The samples were fixed according to Chamberlain. They were coated with gelatin by the standard method and leaf cross-sections (each 10–15 microns in width) were made with the help of a freezing microtome. The sections were stained with safranin. Also the prints of leaves’ epidermis from the adaxial and abaxial leaf surfaces in the daytime were performed. The microscopic measurements were carried out using an eyepiece-micrometer on microscope XSP-146TR. The obtained data were statistically processed with the help of program Statistica 6 on P≤0,05 confidence level. The pictures were taken with the digital camera Canon Power Shot A630.

RESULTS AND DISCUSSIONs Studying biological peculiarities of N. gigantea it was determined that they are spread in lakes and in marshes of Australian tropics, and also they are spread on the North of Arnhemland peninsula. The climate in natural grow of species is tropical monsoon. This is the region of moisture tropical forests, where annually falls 2000- 4000 mm of atmospheric precipitations with equal distribution. The air temperature is +25–30ºС. The difference between the cold and warm season temperatures does not exceed +1–6ºС. The type of the river regulation is amurensis, that is characterized by the advantage of summer flowing and averages 85–95% per year. Winters are dry and summers are hot but with strong rains. Nymphaea gigantea is a perennial herbal tuberiform plant. The underground shoots are elongated, the tubers are (8±0.2) cm long, and (5±0.2) cm wide. The leaves are orbicular, with crenate margin (80±0.5) cm long, and (70±0.5) cm wide. The above-water leaves are emerald and under the water they are marble-pink, with noticeable venation. The leaves are crimson along the edges and have sharp edges for (5±0.2) cm long, the petioles are 0.15–2 m long and have olive colour. In nature the tuberiform plants of the Nymphaea genus including N. gigantea are Rev. Bot., Vol. VI, Nr. 1(8), Chişinău, 2014 105 flowering and fruit bearing. They are developing in spring, during a short period of rains. The ground-based part of vegetative and generative organs die off with the end of rains and the plants spend a bigger part of the year at a tuberous condition in moist mud, like a simple ephemeroides- geophites. The renewal of leaves and roots is observed in parent tubers and child tubers and in ascending stolons only during the rain periods or when water-level is raising. During long flood periods the plants can completely die off and then in 4–5 years they can gradually be renewed from seeds [5; 14]. In conditions of protected soil of Botanical garden we have observed the conditions of ontogenesis periods of N. gigantea. In natural conditions of tropical rivers of Australia these periods are limited by water level abatement (from October till December) and flood (February-March). That is why seeds grow up only next year. Periods and conditions of ontogenesis of the N. gigantea Hook Latent period: Condition “seed” (sm) is a period of seeds ripening which is observed in spring, after 14–18 days of growing up in temperature of +18°–20°C. Such conditions for them we create in April. The type of growing up is underwater; they grow up in upper layers of soil with a depth of water layer of 5–8 cm. This condition lasts for 8–9 months. Pregenerative period: Condition “sprout” (pl) – the sprouts are with awl-shaped leaf; they stay in this condition for 14 days. The condition of “juvenile plants” (j) is characterized by the appearance of one submerged elongated and wide spear-shaped leaf and 5–6 elongated and round leaves and bunches of 10–15 roots (this period lasts for a year). The condition of “immature plants” (im) – is characterized by appearance of 3–5 floating orbicular leaves with crenate margin, and their dying closer to winter. This condition is characterized by forming of an underground sprout – a circular tuber of pink-violet colour and bunches of 20–30 roots, which disappear during a relative rest period and grow back in April (this period lasts 2–3 years). The period of “virginal plant” (v) is characterized by continuation of forming of underground sprouts. It’s also characterized by forming plant tubers, which enlarge and become a violet-brown colour. In April 5–8 elongated-circular underwater leaves appear, and in May the leaves are floating, orbicular with crenate margin which have 20–30 cm in diameter. By winter these leaves die off and an underground sprout which is a circular tuber of a pink-violet colour and a bunch of 20–30 roots are formed. They die off during a relative rest period in November and grow back in April (it lasts for 3–4 years).

Generative period: Condition of “young generative plants” (g-1) is characteri­ zed by appearance in May of 15–25 floating orbicular with crenate margin leaves which have 80–85 sm (have 3–4 years old plants), a bunch of 50–60 roots forms. It lasts for 40–45 days after relative rest period. Condition of “middle aged plant”

(g 2) is characterized by appearance of 15–25 floating orbicular with crenate margin leaves in June which have 80–85 cm in diameter and an underwater bud which starts flowering (in 15–16 days). The appearance of flowers is cyclically connected with the 106 Rev. Bot., Vol. VI, Nr. 1(8), Chişinău, 2014 appearance of a floating leaf. The plants flower for 8–9 days, from June till October. Flowering starts in the morning hours from 8 a.m. till 6–7 a.m. During a day the flowers are opened for 12–13 hours, they are closed for 10–11 hours. It was studied, that a strict cyclic recurrence between petals and anthers is absent. Fruit is bacciform. The ripening period is 20–25 days. The seed has an oval shape, with an arillus, a round top is (5±0.2) cm long, and (3±0.2) cm wide. The arillus destroys in 5–6 hours after ripening of a fruit. The spermoderm is coriaceous, longitudinal-striated. The fruit become fully ripe in 25–30 days after an artificial fertilizing of a first-day flowering flower. Weight of 1,000 seeds is 14.6–16.5 grams. One fruit contains 550–600 seeds. Seeds’ mass and their quantity have a negative correlation; the mass is changes inversely to the quantity [6]. The plants stay in this condition for 5–6 months.

Condition “old generative plants” (g-3) is determined by the end of flowering (in October-November), by gradual disappearing of floating leaves. An underground sprout (formed tubes) deepens in the thickness of soil with the help of contractile roots. Then an underground sprout and roots die off (it lasts for 6–9 weeks). Postgenerative period: Condition “subsenile plants” (ss) is characterized by accumulation of nutrients in tubers, by their enlargement up to 8–10 cm in length, and 5-7 cm in width, and by change of colour to dark violet-brown (it lasts for 40– 54 months). Condition “senile plants” (s) is characterized by formation of vertical and horizontal stoles on a parent tuber and also by formation of child tubers (it lasts for 2–3 years). Condition “dying plants” (sc) is observed on parent tubers which become hollow and float up to the surface of the water or rot in the thickness of soil (it lasts for 1,5–2 years). The anatomical studies have shown that the leaves of considered species (located on the water and above the water) are epistomatous. They have stomata of anomocytic type (fig. 1A). The epidermis cells have flexuous shape and have no trichomes. The projection of the epidermal cells area is elongated. The upper epidermis is thicker than the lower one (16.66±0.3 and 18.74±4.17 μm respectively), although a cuticle and wax are almost absent on the lower epidermis. A palisade mesophyll consists of two- tree layers of cells, its thickness is 131.2±10.48 μm. It also contains large intercellular spaces filled with the air which are not typical for other species of the Nymphaea genus (fig. 1B). A spongy mesophyll is presented by aerenchyma (its thickness is 149.94±24.52 μm). Conductive tissue is developed poorly, that is typical for all aquatic plants. Mechanical tissue is represented by asterosclereids with calciumoxalate crystals frequent in cell walls of mesophyll. In the studied plants the stomata were found only on adaxial surface of floating and above-water leaves. The stomata are absent on abaxial surface of leaves of both types. There are epidermis cells which are bigger with less sinuous cell walls. There are hydropots on abaxial surface of leaves (the water stomata with the thickened walls Rev. Bot., Vol. VI, Nr. 1(8), Chişinău, 2014 107 are often adjacent to a large air cavity, surrounded by radially placed epidermal cells) that are involved in the process of the excretive of excess water and mineral salts. The hydropots are larger in size and they are located with less density as compared to stomata on adaxial surface (Table 1). Table 1 Morphometric parameters of stoma and hydropot of Nymphaea gigantea Hook Amount Amount of of stomata Length of Width of hydropots Length of Width of pcs. / 1 stomata µm stomata, pcs. / 1 hydropots µm hydropots, mm2 µm mm2 µm Floating 337±49 23.95±1.1 21.33±1.9 90±30 31.28±3.20 29.21±2.5 Above- water 225±45* 23.14±2.4 19.5±2.3* 97±34 30.39±2 26.8±2.4* • – P≤0,05 compared with this parameter of floating leaves. Comparing floating and above-water leaves, it can be observed the reduction of the number of stomata per unit of area. And smaller sizes of stomata and hydropots also can be observed in above-water leaves. These parameters testify to a decrease in the necessity for excretive of excess water and mineral salts by above-water leaves.

Fig. 1. Micrographs Nymphaea gigantea Hook.: А) upper epidermis, B) cross section of lamina: 1 – upper epidermis, 2 – palisade mesophyll, 3 – aerenchyma, 4 – lower epidermis, 5 – hydropot, 6 – stoma, 7 – asterosclereida

CONCLUSIONS According to the complex researches the development of N. gigantea in greenhouse conditions continues for 8–10 years. It takes 3–4 years of development from sprouting seeds to flowering plants. After that, a phase of flowering and fruit bearing begins that lasts for 5–6 months. For the next 2–3 years the plants continue their flowering and bearing fruit. During the next two years the plants continue their flowering but less abundantly. It was established that the flower of N. gigantea isn’t 108 Rev. Bot., Vol. VI, Nr. 1(8), Chişinău, 2014 airhaic, and its special structure proves the existence in its structure of cylinders on which the sepals, petals, anthers and a pistil are accommodated. A parent tuber dies off gradually after forming child tubers. It was proved that N. gigantea relates to a tuberous biomorphological structure. It was proved that the leaves of N. gigantea plants have specific anatomical features except those features that are typical for the plants of N. gigantea genus. It was founded that except an expressed aerenhim, with single asterosclereids and hydropots on abaxial surface, there are large intercellular spaces filled with the air in a palisade mesophyll. The heterophylia is observed annually on a morphological level after a relative rest period.

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